Chromatin Structure and the Cell Cycle

Pancreatic DNase I is used to probe the structure of chromatin isolated from synchronized HeLa cells. The degree to which DNA in chromatin is protected from DNase attack varies during the G(1), S, and G(2) phases of the cell cycle. In addition, the DNase sensitivity of chromatin from contact-inhibited African green monkey kidney cells differs from that of actively dividing, subconfluent cultures. These cell cycle-dependent chromatin changes were observed consistently at all enzyme concentrations (5000-fold range) and incubation times (15 min-2 hr) tested. The results indicate that the degree of complexing between DNA and chromosomal proteins changes during interphase, and they suggest that the chromosome coiling cycle of visible mitosis may extend in more subtle form over the entire cell cycle.     

Nucleosomes associated with newly replicated DNA have an altered conformation

In vitro DNA synthesis was studied in HeLa cell nuclei, with emphasis on the question of whether newly replicated DNA is associated with nucleosomes. The newly replicated DNA was twice as sensitive to digestion by micrococcal nuclease as mature chromatin DNA, reaching a limit digest at 20-25% acid-insoluble product. Examination of the intermediates of digestion by micrococcal nuclease showed the nuclease-resistant, new DNA to be complexed in nucleosomes. However, structural differences were evident at both the polynucleosomal and the core particle level. The nucleosomes on newly replicated DNA were arranged with a repeat size of 165-170 base pairs-i.e., smaller than the 185-base-pair repeat of mature chromatin. The heterogeneity of polynucleosomal multimers, evident in digests of whole chromatin, was reduced in newly replicated chromatin such that the multimers resolved as sharply defined bands. Nucleosomal core particles associated with newly replicated DNA had a different conformation from particles in mature chromatin based on the following lines of evidence: (i) during micrococcal nuclease digestion, the monomer nucleosomes did not accumulate but were rapidly degraded under certain conditions; (ii) micrococcal nuclease limit digest patterns and DNase I digestion patterns, both of which reflect internal nucleosomal protein DNA associations, differed significantly from control patterns. These findings bear directly on models postulated for nucleosome-DNA interactions during chromation replication. A possible mechanism to account for the conformational change and its role in replication are discussed.     

Organization of spacer DNA in chromatin.

Detailed analysis of the DNA fragment patterns produced by DNase I digestion of yeast, HeLa, and chicken erythrocyte nuclei reveals surprising features of nucleosome phasing. First, the spacer regions in phased yeast chromatin must be of lengths (10m + 5) base pairs, where m = 0, 1, 2,....This feature is not seen in parallel studies of chicken erythrocyte chromatin. The 5-base pair increment in the yeast spacer imposes interesting restraints on the higher order structure of yeast chromatin. Second, we have been able to simulate the DNase I cutting patterns and get good agreement with the observed yeast patterns. Third, three different chromatins show a long range periodicity in the DNase I digest pattern, with a period half that of the staphylococcal nuclease repeat. These results suggest that the amount of chromatin observed in discrete extended-ladder bands is a minimum estimate of phasing and in fact phasing may be a more general feature.     

Hexamethylenebisacetamide-resistant murine erythroleukemia cells have altered patterns of inducer-mediated chromatin changes.

We determined inducer-mediated changes in chromatin structure near the globin genes in a variant line of murine erythroleukemia cells (MELC). The variant cell line, R1, was derived from the inducer-sensitive DS19 cell line by selection for inducer-resistance. R1 cells are resistant to induction of erythroid differentiation by hexamethylenebisacetamide (HMBA) whereas the parental line is HMBA-sensitive. Uninduced MELC (both inducer-sensitive DS19 cells and inducer-resistant R1 cells) have DNase I-sensitive sites in chromatin containing the alpha 1- and beta maj-globin genes. These nuclease-sensitive regions are located within the beta maj-globin second intervening sequence (IVS2) and near the alpha 1-globin gene 5' cap site. Culture with HMBA causes changes in chromatin structure in both parental and variant cell lines. In DS19 cells, the DNase I-sensitive site within the beta maj-globin IVS2 becomes more resistant to nuclease cleavage, and a new DNase I-sensitive region develops near the beta maj-globin cap site. In addition, the nuclease-sensitive region adjacent to the cap site of the alpha 1-globin gene increases, and a novel 5' nuclease-sensitive site is also established. In R1 cells, HMBA-mediated changes in chromatin structure are incomplete. The DNase I-sensitive site within the beta maj-globin IVS2 becomes more resistant to nuclease cleavage, but the nuclease sensitivity near the beta maj-globin cap site does not increase to the extent observed in DS19 cells. The pattern of nuclease sensitivity near the alpha 1-globin gene is essentially unchanged after culture of R1 cells with HMBA. Thus, in R1 cells, resistance to HMBA-induced expression of globin genes is associated with failure to detect inducer-mediated changes in chromatin structure 5' to the cap site of the alpha 1- and beta maj-globin genes. These results also suggest that the increased nuclease resistance of a site in the beta maj-globin IVS2 does not depend on the establishment of a DNase I-sensitive region near the beta maj-globin gene cap site.     

Reconstitution of hyperacetylated, DNase I-sensitive chromatin characterized by high conformational flexibility of nucleosomal DNA

Increased acetylation at specific N-terminal lysines of core histones is a hallmark of active chromatin in vivo, yet the structural consequences of acetylation leading to increased gene activity are only poorly defined. We employed a new approach to characterize the effects of histone acetylation: A Drosophila embryo-derived cell-free system for chromatin reconstitution under physiological conditions was programmed with exogenous histones to assemble hyperacetylated or matching control chromatin of high complexity. Hyperacetylated chromatin resembled unmodified chromatin at similar nucleosome density with respect to its sensitivity toward microccal nuclease, its nucleosomal repeat length, and the incorporation of the linker histone H1. In contrast, DNA in acetylated chromatin showed an increased sensitivity toward DNase I and a surprisingly high degree of conformational flexibility upon temperature shift pointing to profound alterations of DNA/histone interactions. This successful reconstitution of accessible and flexible chromatin outside of a nucleus paves the way for a thorough analysis of the causal relationship between histone acetylation and gene function.     

Differential scanning calorimetry of nuclei reveals the loss of major structural features in chromatin by brief nuclease treatment.

Differential scanning calorimetry revealed that chromatin melts in four distinct transitions in intact HeLa nuclei at 60 degrees C, 76 degrees C, 88 degrees C, and 105 degrees C. Calorimetry of whole cells was characterized by the same four transitions along with another at 65 degrees C, which was probably RNA. Isolated chromatin, however, melted in only two transitions at 72 degrees C and 85 degrees C. Very brief digestion of HeLa nuclei with either micrococcal nuclease or DNase I resulted in the conversion of a structure that melted at 105 degrees C to one that melted at 88 degrees C. Further digestion with micrococcal nuclease to the level of the mononucleosome did not result in any further substantial changes in either enthalpy or melting temperatures. In contrast, further DNase I digestion that resulted in cleavage within the nucleosome produced a pronounced shift in melting temperatures to broad transitions at 62 degrees C and 78 degrees C.     

Cleavage of chromatin with methidiumpropyl-EDTA . iron(II).

Methidiumpropyl-EDTA . iron(II) [MPE . Fe (II)] cleaves double-helical DNA with considerably lower sequence specificity than micrococcal nuclease. Moreover, digestions with MPE . Fe(II) can be performed in the presence of certain metal chelators, which will minimize the action of many endogenous nucleases. Because of these properties MPE . Fe(II) would appear to be a superior tool for probing chromatin structure. We have compared the patterns generated from the 1.688 g/cm3 complex satellite, 5S ribosomal RNA, and histone gene sequences of Drosophila melanogaster chromatin and protein-free DNA by MPE . Fe(II) and micrococcal nuclease cleavage. MPE . Fe(II) at low concentrations recognizes the nucleosome array, efficiently introducing a regular series of single-stranded (and some double-stranded) cleavages in chromatin DNA. Subsequent S1 nuclease digestion of the purified DNA produces a typical extended oligonucleosome pattern, with a repeating unit of ca. 190 base pairs. Under suitable conditions, relatively little other nicking is observed. Unlike micrococcal nuclease, which has a noticeable sequence preference in introducing cleavages, MPE . Fe(II) cleaves protein-free tandemly repetitive satellite and 5S DNA sequences in a near-random fashion. The spacing of cleavage sites in chromatin, however, bears a direct relationship to the length of the respective sequence repeats. In the case of the histone gene sequences a faint, but detectable, MPE . Fe(II) cleavage pattern is observed on DNA, in some regions similar to and in some regions different from the strong chromatin-specified pattern. The results indicate that MPE . Fe(II) will be very useful in the analysis of chromatin structure.     

Nucleosome segregation at a defined mammalian chromosomal site.

When animal cells replicate chromatin under conditions precluding new histone biosynthesis, half of the daughter DNAs are devoid of nucleosomes and are sensitive to staphylococcal nuclease. DNA sequences resistant to nuclease are associated with preexisting nucleosomes, which redistribute to progeny DNA duplexes during replication. We labeled newly replicated DNA sequences in a simian virus 40 (SV40)-transformed Chinese hamster cell clone with 5-bromodeoxyuridine (BrdUrd) in the presence and absence of a protein biosynthesis inhibitor, emetine. We resolved single-stranded BrdUrd- and dT-DNA sequences protected from nuclease digestion by nucleosomes and determined from which strands of the integrated viral DNA parental template (dT) and newly replicated progeny (BrdUrd) sequences were derived. Because we knew that the cell clone studied contained all of its integrated SV40 DNA at a single chromosomal site, we were able to determine that preexisting nucleosomes segregated to only one of the two daughter duplexes containing the integrated viral sequence. Additionally, in the presence of emetine, the integrated viral origin of replication, ORIsv, appeared not to function as a chromosomal replication origin, perhaps reflecting the drug's effect on synthesis of SV40 large tumor antigen.     

Isolation of a subclass of nuclear proteins responsible for conferring a DNase I-sensitive structure on globin chromatin.

The globin gene is preferentially sensitive to digestion by DNase I in erythrocyte chromatin but not in brain, fibroblast, or oviduct chromatin. Elution of the erythrocyte chromatin with 0.35 M NaCl leads to no detectable change in the gross structure of individual nucleosomes; however, in this depleted chromatin the globin gene is no longer preferentially sensitive to DNase I. Reconstitution of the depleted chromatin with either the entire 0.35 M NaCl fraction or a subclass from this fraction greatly enriched in two high mobility group proteins (nos. 14 and 17) results in the successful reconstitution of DNase I sensitivity of the globin gene. For all of these preparations, the inactive ovalbumin gene exhibited no preferential sensitivity to DNase I. Reconstitution of the erythrocyte 0.35 M NaCl fraction with depleted brain chromatin resulted in no preferential sensitivity of the globin gene in brain chromatin; however, reconstitution of the brain 0.35 M NaCl fraction with depleted erythrocyte chromatin led to successful reconstitution of DNase I sensitivity of the globin gene. Thus, the eluted proteins responsible for conferring DNase I sensitivity are probably not tissue-specific and probably do not recognize specific DNA sequences.     

XMCM7, a novel member of the Xenopus MCM family, interacts with XMCM3 and colocalizes with it throughout replication.

A minichromosome maintenance (MCM) protein complex has been implicated in restricting DNA replication to once per cell cycle in Xenopus egg extracts, based on the behavior of a single protein, XMCM3. Using a two-hybrid screen with XMCM3, we have identified a novel member of the MCM family in Xenopus that is essential for DNA replication. The protein shows strong homology to Saccharomyces cerevisiae MCM7 (CDC47) and has thus been named XMCM7. XMCM7 is present in a multiprotein complex with other MCM proteins. It binds to chromatin and is displaced from chromatin by the act of replication. XMCM7 does not preferentially colocalize with sites of DNA replication but colocalizes with XMCM3 throughout replication. Immunodepletion of the MCM complex from Xenopus egg extract by anti-XMCM7 antibodies inhibits DNA replication of sperm and permeable HeLa G2 nuclei but not permeable HeLa G1 nuclei. Replication capacity of the Xenopus egg extract immunodepleted of the MCM complex by anti-XMCM7 antibody can be rescued by MCM proteins eluted from anti-XMCM3 antibody. We conclude that both proteins are present in the same complex in Xenopus egg extract throughout the cell cycle, that they remain together after binding to chromatin and during DNA replication, and that they perform similar functions.